Attenuated GJIC In Endometrial Stromal Cells Involvement In The Pathogenesis Of Endometriosis And The Regulation Of GJIC In Stromal Cells From Endometriotic Lesions And Endometria With Endometriosis By ATRA

Endometriosis (EMs) is a common gynecological disorder, with an incidence of 10-15% in reproductive aged women. The pathogenesis of endometriosis remains unclear, and current therapeutic effects are not satisfied. New strategies against EMs are urgently needed.Similar to tumors'biological behaviors, EMs has the nature of adhesion, invasion and metastasis, which suggests that EMs and some tumors share the same mechanisms of pathogenesis. The dysfunction of gap junctional intercellular communication (GJIC) occurred in cancer cells, suggesting that aberrant GJIC might involve in the development of EMs.GJIC is the main way for adjacent cells to communicate and transmit signal, which is necessary for maintenance of tissue homeostasis. Functional GJIC is mediated by connexons consisting of proteinaceous cylinders with a hydrophilic channel. The channels allow ions (Na+, K+, Ca2+, etc) and small molecules (bellow 1000Da, such as cAMP, cGMP, IP3) to travel freely. Each connexon, structural units of GJIC, was composed of a hexamer of connexin (Cx). There are three Cxs, Cx43, Cx32, Cx26, expressing in rodents and human endometrium.Endometrial stromal cells may play an important role in the development of EMs. Normal stromal cells are with consummate function of GJIC, and they can modulate the status of GJIC in endometrial epithelial cells by gap junction.All-trans-retinoic acid (ATRA) can enhance Cx expression and up-regulate GJIC in many tumor cells, even induce the differentiation of cancers. ATRA can also up-regulate functional GJIC in normal stromal cells, but it is still ambiguity whether ATRA can modulate Cx expression and GJIC in endometrial stromal cells from patients with EMs.This study composed of three sections: (1) Stromal cells and epithelial cells were isolated from the ectopic endometrial lesions located in ovaries, eutopic endometria from women with EMs and normal endometria. (2) Endometrial stromal cells and epithelial cells ware cultured in vitro to explore the expression of Cx and functional GJIC. Antisense oligodeoxyribonucleotides (ASODNs) was used to suppress Cx expression in normal stomal cells. Cell growth, adhesion and invasion were investigated in normal stromal cells after treatment with ASODNs. The correlation between deficient GJIC in endometrial stromal cells and pathogenesis of EMs was analyzed. (3) ATRA was used to modulate the function of GJIC in stromal cells from EMs. The objective was to determine whether ATRA be an agent to cure EMs.PARTⅠIN VITRO CULTURE OF ENDOMETRIAL STROMAL AND EPITHELIAL CELLS FROM EMS AND NORMAL ENDOMETRIUMObjective: To establish an approach to purify and culture human endometrial stromal and epithelial cells from endometriotic lesions loctated in ovaries, eutopic endometria with EMs and normal endometria, respectively.Methods: The cases were randomly chosen from patients hospitalized receiving surgical interventions in Feb 2006-Jan 2007. Samples from eutopic endometria or endometriotic lesions were collected from 41 cases suffered from endometriosis, in all, 24 cases were accompanied with unilateral or bilateral chocolate cysts in ovaries. 30 cases received hysterectomy for uterine myoma were recruited as control. Endometrial tissue near the bottom of the uterus was scraped, and the lining of chocolate cyst located in ovary was striped. Digestion, purification and culture of endometrial stromal and epithelial cells were performed.Results: The success rate of isolation and culture was 33.3% (8/24) of endometriotic stromal and epithelial cells, 92.7% (38/41) of eutopic endometrial stromal and epithelial cells with EMs, and 93.3% (28/30) of normal endometrial stromal and epithelial cells. The purities of eutopic stromal and epithelial cells from endometrium with or without EMs were 98% and 95% respectively. The purities of ectopic stromal and epithelial cells were 95% and 92% respectively. Both stromal cells and epithelial cells could be cryopreservated and thawed successfully.Conclusion: The higher yields and viabilities were realized through this multistep procedure.PARTⅡTHE EXPRESSION OF CONNEXIN AND GJIC IN ENDOMETRIAL STROMAL CELLS IN THE DEVELOPMENT OF ENDOMETRIOSISObjective: To investigate the different expression of Cx and functional GJIC in stromal cells in endometriotic lesion and eutopic endometrium with EMs from those of in normal endometrium, and to reveal the correlation between deficient GJIC in endometrial stromal cells and the pathogenesis of EMs.Methods: (1) The immumofluorescence assay manifested the expression of Cx43, Cx32 and Cx26 protein in stromal and epithelial cells from endometriotic lesions, eutopic endometria with EMs and normal endometria. (2) Fluorescence recovery after photobleaching (FRAP) was applied to analyze the function of GJIC in stromal and epithelial cells with and without EMs. Furthermore, GJIC status in epithelial cells regulated by different stromal cells was evaluated in co-culture system in vitro. (3) The abilities of growth, adhesion and invasion were investigated in normal stromal cells after downregulating Cx43 with antisense oligodeoxyribonucleotides (ASODNs).Results: The level of Cx43 in endometrial stromal cells was gradually decreased from normal endometrium, eutopic endometrium with EMs to endometriotic tissue. Cx32 and Cx26 protein were observed in normal endometrial epithelial cells and eutopic epithelial cells from endometrium with EMs. There was no difference of Cx32 protein expression in epithelial cells between normal endometrium and eutopic endometrium with EMs, nor did Cx26 protein. Cx43 was aberrant expression in endometriotic epithelial cells. There was similar functional GJIC status in epithelial cells from control, eutopic endometrium with EMs and endometriotic lesion. However, the function of GJIC in stromal cells from ectopic endometrial tissues was lower than those from the other two groups, the highest functional GJIC was observed in normal endometrial stromal cells, and the distinctions among these groups were significant. The ability of regulation of GJIC in epithelial cells by stromal cells from endometriotic lesion or eutopic endometrium with EMs was weaker compared with those stromal cells from control group. Stromal cells'GJIC and ability to modulate epithelial cells were closely related to their Cx43 protein. Cx43 protein and functional GJIC were suppressed when normal stromal cells were transfected with ASODNs+Lipo, resulting in a faster growth, enhanced adhesion and promoted invasion.Conclusion: The down-regulation of Cx43 and attenuated GJIC were related to the pathogenesis of EMs. Cx43 or GJIC in endometrial stromal cells might be used as potent target for treatment of EMs. PARTⅢMECHANISMS OF REGULATION OF CONNEXIN43 EXPRESSION AND GJIC IN STROMAL CELLS FROM ENDOMETRIOTIC LESIONS AND ENDOMETRIA WITH ENDOMETRIOSIS BY ATRA IN VITROObjective: To explore mechanisms involving in the regulation of GJIC by ATRA in stromal cells from endometriotic lesions and eutopic endometria with EMs.Methods: (1) Stromal cells from endometriotic lesions and eutopic endometria with EMs were treated with ATRA with concentrations of 0.1μmol/L, 1μmol/L and 10μmol/L for 24, 48, 72, 96 and 120 h, respectively, Laser scanning confocal microscope was applied to determine the function of GJIC in cells. The mRNA and protein levels of Cx43 were investigated. (2) ICI 182 780, an estrogen receptor downregulator, was introduced to analyze the relationship between the effects of ATRA and estrogen receptor. (3) An inductor, 12-o-tetradecanoylphorbol-13-acetate (TPA), was used to judge that the dephosphorylated form or phosphorylated species of Cx43 would contribute to the up-regulation of functional GJIC in stromal cells.Results: GJIC was enhanced significantly either in ectopic stromal cell or eutopic stromal cell from endometrium with EMs after being exposed to 1μmol/L and 10μmol/L ATRA. The up-regulation persisted up to 72h. No evidence for enhanced GJIC was confirmed in these stromal cells treatment with 0.1μmol/L ATRA. Treatment of stromal cells with 1μmol/L ATRA up regulated the expressions of Cx43 mRNA and protein. TPA inhibited the effect of ATRA on modulation of functional GJIC in ectopic stromal cells and eutopic stromal cells from endometrium with EMs. However, ICI 182 780 didn't have the inhibitory effect similar to TPA.Conclusions: GJIC in ectopic stromal cells and eutopic stromal cells from endometrium with EMs was up-regulated by ATRA in a manner of time- and dosedependent. ATRA enhanced Cx43 expression on both gene and protein levels, and induced or maintained the dephosphorylated form of Cx43 protein in these stromal cells.